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1.  Mechanism of inhibition of Mycobacterium tuberculosis antigen 85 by ebselen 
Nature communications  2013;4:2748.
The increasing prevalence of drug-resistant tuberculosis highlights the need for identifying new antitubercular drugs that can treat these infections. The antigen 85 (Ag85) complex has emerged as an intriguing mycobacterial drug target due to its central role in synthesizing major components of the inner and outer leaflets of the mycobacterial outer membrane. Here we identify ebselen as a potent inhibitor of the Mycobacterium tuberculosis Ag85 complex. Mass spectrometry data show that ebselen binds covalently to a cysteine residue (C209) located near the Ag85C active site. The crystal structure of Ag85C in the presence of ebselen shows that C209 modification restructures the active site, thereby disrupting the hydrogen-bonded network within the active site that is essential for enzymatic activity. C209 mutations display marked decreases in enzymatic activity. These data suggest that compounds using this mechanism of action will strongly inhibit the Ag85 complex and minimize the selection of drug resistance.
doi:10.1038/ncomms3748
PMCID: PMC4049535  PMID: 24193546
2.  Subfractionation and analysis of the cell envelope (lipo)polysaccharides of Mycobacterium tuberculosis 
The cell envelope of Mycobacterium tuberculosis, the causative agent of tuberculosis in humans, is the source of carbohydrates of exceptional structure which play essential roles in the physiology of the bacterium and in its interactions with the host during infection. Much of what is known about their biosynthesis was derived from the phenotypic analysis of knock-out or conditional knock-out mutants of Mycobacteria generated by random or specific insertional mutagenesis. Here, we describe the current techniques used to subfractionate M. tuberculosis cells and investigate major quantitative and qualitative changes in their cell envelope (lipo)polysaccharides.
doi:10.1007/978-1-62703-245-2_19
PMCID: PMC4041545  PMID: 23299743
Mycobacterium tuberculosis; arabinogalactan; lipoarabinomannan; lipomannan; glucan; capsule
3.  Inactivation of Fructose-1,6-Bisphosphate Aldolase Prevents Optimal Co-catabolism of Glycolytic and Gluconeogenic Carbon Substrates in Mycobacterium tuberculosis 
PLoS Pathogens  2014;10(5):e1004144.
Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.
Author Summary
The development of new chemotherapies targeting Mycobacterium tuberculosis (Mtb) will benefit from genetic evaluation of potential drug targets and a better understanding of the pathways required by Mtb to establish and maintain chronic infections. We employed a genetic approach to investigate the essentiality of fructose-1,6-bisphosphate aldolase (FBA) for growth and survival of Mtb in vitro and in mice. A conditional fba mutant revealed that Mtb requires FBA for growth in the acute phase and for survival in the chronic phase of mouse infections. In vitro essentiality of fba was strictly condition-dependent. An FBA deletion mutant (Δfba) required a balanced combination of carbon substrates entering metabolism above and below the FBA-catalyzed reaction for growth and died in media with single carbon sources and in mouse lungs. Death of Δfba in vitro was associated with the perturbation of intracellular metabolites. These studies highlight how a conditional fba mutant helped identify conditions in which FBA is dispensable for growth of Mtb, evaluate FBA as a potential target for eliminating persistent bacilli and offer insight into metabolic regulation of carbon co-catabolism in Mtb.
doi:10.1371/journal.ppat.1004144
PMCID: PMC4031216  PMID: 24851864
4.  Design, Synthesis and Anti-tuberculosis Activity of 1-Adamantyl-3-heteroaryl Ureas with Improved in vitro Pharmacokinetic Properties 
Bioorganic & medicinal chemistry  2013;21(9):2587-2599.
Out of the prominent global ailments, tuberculosis (TB) is still one of the leading causes of death worldwide due to infectious disease. Development of new drugs that shorten the current tuberculosis treatment time and have activity against drug resistant strains is of utmost importance. Towards these goals we have focused our efforts on developing novel anti-TB compounds with the general structure of 1-adamantyl-3-phenyl urea. This series is active against Mycobacteria and previous lead compounds were found to inhibit the membrane transporter MmpL3, the protein responsible for mycolic acid transport across the plasma membrane. However, these compounds suffered from poor in vitro pharmacokinetic (PK) profiles and they have a similar structure/SAR to inhibitors of human soluble epoxide hydrolase (SEH) enzymes. Therefore, in this study the further optimization of this compound class was driven by three factors: 1) to increase selectivity for anti-TB activity over human sEH activity, 2) to optimize PK profiles including solubility and 3) to maintain target inhibition. A new series of 1-adamantyl-3-heteroaryl ureas was designed and synthesized replacing the phenyl substituent of the original series with pyridines, pyrimidines, triazines, oxazoles, isoxazoles, oxadiazoles and pyrazoles. This study produced lead oxadiazole and pyrazole substituted adamantyl ureas with improved in vitro PK profiles, increased selectivity and good anti-TB potencies with sub µg/mL minimum inhibitory concentrations.
doi:10.1016/j.bmc.2013.02.028
PMCID: PMC3780361  PMID: 23498915
5.  Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis 
Future microbiology  2013;8(7):10.2217/fmb.13.52.
Most of the newly discovered compounds showing promise for the treatment of TB, notably multidrug-resistant TB, inhibit aspects of Mycobacterium tuberculosis cell envelope metabolism. This review reflects on the evolution of the knowledge that many of the front-line and emerging products inhibit aspects of cell envelope metabolism and in the process are bactericidal not only against actively replicating M. tuberculosis, but contrary to earlier impressions, are effective against latent forms of the disease. While mycolic acid and arabinogalactan synthesis are still primary targets of existing and new drugs, peptidoglycan synthesis, transport mechanisms and the synthesis of the decaprenyl-phosphate carrier lipid all show considerable promise as targets for new products, older drugs and new combinations. The advantages of whole cell- versus target-based screening in the perpetual search for new targets and products to counter multidrug-resistant TB are discussed.
doi:10.2217/fmb.13.52
PMCID: PMC3867987  PMID: 23841633
antibiotic; arabinogalactan; cell envelope; Mycobacterium; mycolic acids; peptidoglycan; tuberculosis
6.  Gene Replacement in Mycobacterium chelonae: Application to the Construction of Porin Knock-Out Mutants 
PLoS ONE  2014;9(4):e94951.
Mycobacterium chelonae is a rapidly growing mycobacterial opportunistic pathogen closely related to Mycobacterium abscessus that causes cornea, skin and soft tissue infections in humans. Although M. chelonae and the emerging mycobacterial pathogen M. abscessus have long been considered to belong to the same species, these two microorganisms considerably differ in terms of optimum growth temperature, drug susceptibility, pathogenicity and the types of infection they cause. The whole genome sequencing of clinical isolates of M. chelonae and M. abscessus is opening the way to comparative studies aimed at understanding the biology of these pathogens and elucidating the molecular bases of their pathogenicity and biocide resistance. Key to the validation of the numerous hypotheses that this approach will raise, however, is the availability of genetic tools allowing for the expression and targeted mutagenesis of genes in these species. While homologous recombination systems have recently been described for M. abscessus, genetic tools are lacking for M. chelonae. We here show that two different allelic replacement methods, one based on mycobacteriophage-encoded recombinases and the other on a temperature-sensitive plasmid harboring the counterselectable marker sacB, can be used to efficiently disrupt genes in this species. Knock-out mutants for each of the three porin genes of M. chelonae ATCC 35752 were constructed using both methodologies, one of which displays a significantly reduced glucose uptake rate consistent with decreased porin expression.
doi:10.1371/journal.pone.0094951
PMCID: PMC3989263  PMID: 24739882
7.  High-level Relatedness among Mycobacterium abscessus subsp. massiliense Strains from Widely Separated Outbreaks 
Emerging Infectious Diseases  2014;20(3):364-371.
Three recently sequenced strains isolated from patients during an outbreak of Mycobacterium abscessus subsp. massiliense infections at a cystic fibrosis center in the United States were compared with 6 strains from an outbreak at a cystic fibrosis center in the United Kingdom and worldwide strains. Strains from the 2 cystic fibrosis outbreaks showed high-level relatedness with each other and major-level relatedness with strains that caused soft tissue infections during an epidemic in Brazil. We identified unique single-nucleotide polymorphisms in cystic fibrosis and soft tissue outbreak strains, separate single-nucleotide polymorphisms only in cystic fibrosis outbreak strains, and unique genomic traits for each subset of isolates. Our findings highlight the necessity of identifying M. abscessus to the subspecies level and screening all cystic fibrosis isolates for relatedness to these outbreak strains. We propose 2 diagnostic strategies that use partial sequencing of rpoB and secA1 genes and a multilocus sequence typing protocol.
doi:10.3201/eid2003.131106
PMCID: PMC3944860  PMID: 24565502
Mycobacterium abscessus; Mycobacterium abscessus subsp. massiliense; bacteria; tuberculosis and other mycobacteria; strains; cystic fibrosis; relatedness; outbreaks; geographically distant outbreaks; United States; United Kingdom
8.  Guidance on Management of Asymptomatic Neonates Born to Women With Active Genital Herpes Lesions 
Pediatrics  2013;131(2):e635-e646.
Herpes simplex virus (HSV) infection of the neonate is uncommon, but genital herpes infections in adults are very common. Thus, although treating an infant with neonatal herpes is a relatively rare occurrence, managing infants potentially exposed to HSV at the time of delivery occurs more frequently. The risk of transmitting HSV to an infant during delivery is determined in part by the mother’s previous immunity to HSV. Women with primary genital HSV infections who are shedding HSV at delivery are 10 to 30 times more likely to transmit the virus to their newborn infants than are women with recurrent HSV infection who are shedding virus at delivery. With the availability of commercial serological tests that reliably can distinguish type-specific HSV antibodies, it is now possible to determine the type of maternal infection and, thus, further refine management of infants delivered to women who have active genital HSV lesions. The management algorithm presented herein uses both serological and virological studies to determine the risk of HSV transmission to the neonate who is delivered to a mother with active herpetic genital lesions and tailors management accordingly. The algorithm does not address the approach to asymptomatic neonates delivered to women with a history of genital herpes but no active lesions at delivery.
doi:10.1542/peds.2012-3216
PMCID: PMC3557411  PMID: 23359576
newborn; herpes simplex virus; acyclovir; pregnancy
9.  Feasibility of a Web-Based Survey of Hallucinations and Assessment of Visual Function in Patients With Parkinson’s Disease 
Background
Patients with Parkinson’s disease (PD) experience visual hallucinations, which may be related to decreased contrast sensitivity (ie, the ability to discern shades of grey).
Objective
The objective of this study was to investigate if an online research platform can be used to survey patients with Parkinson’s disease regarding visual hallucinations, and also be used to assess visual contrast perception.
Methods
From the online patient community, PatientsLikeMe, 964 members were invited via email to participate in this study. Participants completed a modified version of the University of Miami Parkinson’s disease hallucinations questionnaire and an online vision test.
Results
The study was completed by 27.9% (269/964) of those who were invited: 56.9% of this group had PD (153/269) and 43.1% (116/269) were non-Parkinson’s controls. Hallucinations were reported by 18.3% (28/153) of the Parkinson’s group. Although 10 subjects (9%) in the control group reported experiencing hallucinations, only 2 of them actually described formed hallucinations. Participants with Parkinson’s disease with a mean of 1.75 (SD 0.35) and the control group with a mean of 1.85 (SD 0.36) showed relatively good contrast perception as measured with the online letter test (P=.07). People who reported hallucinations showed contrast sensitivity levels that did not differ from levels shown by people without hallucinations (P=.96), although there was a trend towards lower contrast sensitivity in hallucinators.
Conclusions
Although more Parkinson's responders reported visual hallucinations, a significant number of non-Parkinson's control group responders also reported visual hallucinations. The online survey method may have failed to distinguish between formed hallucinations, which are typical in Parkinson's disease, and non-formed hallucinations that have less diagnostic specificity. Multiple questions outlining the nature of the hallucinations are required. In a clinical interview, the specific nature of the hallucination would be further refined to rule out a vague description that does not indicate a true, formed visual hallucination. Contrary to previous literature, both groups showed relatively good contrast sensitivity, perhaps representing a ceiling effect or limitations of online testing conditions that are difficult to standardize. Steps can be taken in future trials to further standardize online visual function testing, to refine control group parameters and to take steps to rule out confounding variables such as comorbid disease that could be associated with hallucinations. Contacting subjects via an online health social network is a novel, cost-effective method of conducting vision research that allows large numbers of individuals to be contacted quickly, and refinement of questionnaires and visual function testing may allow more robust findings in future research.
doi:10.2196/ijmr.2744
PMCID: PMC3906694  PMID: 24394559
Parkinson’s disease; hallucinations; contrast sensitivity; Charles Bonnet Syndrome
10.  Trends in Adverse Reactions to Trimethoprim-Sulfamethoxazole 
Pediatrics  2013;131(1):e103-e108.
OBJECTIVE:
To examine temporal trends of adverse drug reactions (ADRs) associated with trimethoprim-sulfamethoxazole (TMP-SMX) use in children.
METHODS:
We performed a retrospective observational study to characterize TMP-SMX ADRs in children between 2000 and 2009. We completed a chart review at our institution by identifying children diagnosed with TMP-SMX ADRs. To compare local trends to comparable institutions, we estimated the frequency of hospitalizations for TMP-SMX ADRs at 25 tertiary pediatric hospitals utilizing the Pediatric Health Information System database. To determine whether changes in outpatient prescribing rates occurred, we used the National Ambulatory Medical Care Survey/National Hospital Ambulatory Medical Care Survey.
RESULTS:
At our institution, 109 children were diagnosed with a TMP-SMX ADR (5 cases from 2000 to 2004 as compared with 104 cases from 2005 to 2009). Fifty-eight percent had been treated for a skin and soft tissue infection (SSTI). A similar trend was observed nationally, where the incidence of TMP-SMX ADRs more than doubled from 2004 to 2009 at comparable pediatric hospitals (P < .001). Although national outpatient data revealed no change in overall TMP-SMX prescribing, the percentage of children prescribed TMP-SMX for SSTI sharply increased during the study period (0%–2% [2000-2004]; 9%–17% [2005–2009]).
CONCLUSIONS:
The majority of TMP-SMX ADRs at our institution occurred in conjunction with SSTI treatment. TMP-SMX ADRs have occurred more frequently coincident with increased prescribing for SSTI. Increased usage alone may explain the increasing trend of TMP-SMX ADRs in children; however drug–disease interaction may play a role and requires further investigation.
doi:10.1542/peds.2012-1619
PMCID: PMC3529952  PMID: 23209098
adverse drug reaction; trimethoprim-sulfamethoxazole; skin and soft tissue infections; pediatrics; physician practice patterns
11.  Regulation of Mycolactone, the Mycobacterium ulcerans Toxin, Depends on Nutrient Source 
Background
Mycobacterium ulcerans, a slow-growing environmental bacterium, is the etiologic agent of Buruli ulcer, a necrotic skin disease. Skin lesions are caused by mycolactone, the main virulence factor of M. ulcerans, with dermonecrotic (destruction of the skin and soft tissues) and immunosuppressive activities. This toxin is secreted in vesicles that enhance its biological activities. Nowadays, it is well established that the main reservoir of the bacilli is localized in the aquatic environment where the bacillus may be able to colonize different niches. Here we report that plant polysaccharides stimulate M. ulcerans growth and are implicated in toxin synthesis regulation.
Methodology/Principal Findings
In this study, by selecting various algal components, we have identified plant-specific carbohydrates, particularly glucose polymers, capable of stimulating M. ulcerans growth in vitro. Furthermore, we underscored for the first time culture conditions under which the polyketide toxin mycolactone, the sole virulence factor of M. ulcerans identified to date, is down-regulated. Using a quantitative proteomic approach and analyzing transcript levels by RT-qPCR, we demonstrated that its regulation is not at the transcriptional or translational levels but must involve another type of regulation. M. ulcerans produces membrane vesicles, as other mycobacterial species, in which are the mycolactone is concentrated. By transmission electron microscopy, we observed that the production of vesicles is independent from the toxin production. Concomitant with this observed decrease in mycolactone production, the production of mycobacterial siderophores known as mycobactins was enhanced.
Conclusions/Significance
This work is the first step in the identification of the mechanisms involved in mycolactone regulation and paves the way for the discovery of putative new drug targets in the future.
Author Summary
Mycolactone, a polyketide cytotoxic toxin, is the key virulence factor responsible for large skin ulcers in Buruli ulcer. This disease, mainly occurring in humid tropical zones, especially in West African countries, is due to infection by Mycobacterium ulcerans, a slow-growing environmental mycobacterium. The toxin has destructive effects on the skin, soft tissues and bones. In this study, we brought out for the first time that addition of specific carbohydrate to culture medium induces the down-regulation of the toxin. Furthermore, this decrease in toxin production is correlated with the activation of the iron acquisition pathway, especially by siderophore production. These results show that M. ulcerans adapts its metabolism to culture conditions, which probably reflect its adaptation in its natural habitats. This work is the first step in toxin regulation understanding which is a key issue for a better comprehension of Buruli ulcer physiopathology and the identification of putative therapeutic targets.
doi:10.1371/journal.pntd.0002502
PMCID: PMC3828164  PMID: 24244764
12.  Aldehyde-Resistant Mycobacteria Associated with the Use of Endoscope Reprocessing Systems 
Bacteria can develop resistance to antibiotics, but less is known about their ability to increase resistance to chemical disinfectants. This study randomly sampled three AERs in the USA using aldehydes for endoscope disinfection. Bacterial contamination was found post-disinfection in all AERs and some mycobacteria isolated demonstrated significant resistance to glutaraldehyde and OPA disinfectants. Bacteria can survive aldehyde-based disinfection and may pose a cross-contamination risk to patients.
doi:10.1016/j.ajic.2011.11.004
PMCID: PMC3523318  PMID: 22325730
13.  Cooccurrence of Free-Living Amoebae and Nontuberculous Mycobacteria in Hospital Water Networks, and Preferential Growth of Mycobacterium avium in Acanthamoeba lenticulata 
Applied and Environmental Microbiology  2013;79(10):3185-3192.
The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.
doi:10.1128/AEM.03823-12
PMCID: PMC3685249  PMID: 23475613
14.  Genome Sequence of an Epidemic Isolate of Mycobacterium abscessus subsp. bolletii from Rio de Janeiro, Brazil 
Genome Announcements  2013;1(4):e00617-13.
Multiple isolates of Mycobacterium abscessus subsp. bolletii, collectively called BRA100, were associated with outbreaks of postsurgical skin infections across various regions of Brazil from 2003 to 2009. We announce the draft genome sequence of a newly sequenced BRA100 strain, M. abscessus subsp. bolletii CRM-0020, isolated from a patient in Rio de Janeiro, Brazil.
doi:10.1128/genomeA.00617-13
PMCID: PMC3744681  PMID: 23950125
15.  Temporal Profile of Functional Visual Rehabilitative Outcomes Modulated by Transcranial Direct Current Stimulation (tDCS) 
Objectives
We have previously reported that transcranial direct current stimulation (tDCS) delivered to the occipital cortex enhances visual functional recovery when combined with 3 months of computer-based rehabilitative training in patients with hemianopia. The principal objective of this study was to evaluate the temporal sequence of effects of tDCS on visual recovery as they appear over the course of training and across different indicators of visual function.
Methods
Primary objective outcome measures were i) shifts in visual field border and ii) stimulus detection accuracy within the affected hemifield. These were compared between patients randomized to either vision restoration therapy (VRT) combined with active tDCS or VRT paired with sham tDCS. Training comprised of 2 half hour sessions, 3 times a week for 3 months. Primary outcome measures were collected at baseline (pretest), monthly interim intervals, and at posttest (3 months). As secondary outcome measures, contrast sensitivity and reading performance were collected at pretest and posttest time-points only.
Results
Active tDCS combined with VRT accelerated the recovery of stimulus detection as between-group differences appeared within the first month of training. In contrast, a shift in the visual field border was only evident at posttest (after 3 months of training). TDCS did not affect contrast sensitivity or reading performance.
Conclusions
These results suggest that tDCS may differentially affect the magnitude and sequence of visual recovery in a manner that is task- specific to the type of visual rehabilitative training strategy employed.
doi:10.1111/j.1525-1403.2012.00440.x
PMCID: PMC3389571  PMID: 22376226
transcranial direct current stimulation (tDCS); brain stimulation; hemianopia; visual field; rehabilitation; vision restoration therapy (VRT)
16.  Impact of the deletion of the six mce operons in Mycobacterium smegmatis 
The Mycobacterium smegmatis genome contains six operons designated mce (mammalian cell entry). These operons, which encode membrane and exported proteins, are highly conserved in pathogenic and non-pathogenic mycobacteria. Although the function of the Mce protein family has not yet been established in Mycobacterium smegmatis, the requirement of the mce4 operon for cholesterol utilization and uptake by Mycobacterium tuberculosis has recently been demonstrated. In this study, we report the construction of an M. smegmatis knock-out mutant deficient in the expression of all six mce operons. The consequences of these mutations were studied by analyzing physiological parameters and phenotypic traits. Differences in colony morphology, biofilm formation and aggregation in liquid cultures were observed, indicating that mce operons of M. smegmatis are implicated in the maintenance of the surface properties of the cell. Importantly, the mutant strain showed reduced cholesterol uptake when compared to the parental strain. Further cholesterol uptake studies using single mce mutant strains showed that the mutation of operon mce4 was reponsible for the cholesterol uptake failure detected in the sextuple mce mutant. This finding demonstrates that mce4 operon is involved in cholesterol transport in M. smegmatis.
doi:10.1016/j.micinf.2012.01.007
PMCID: PMC3615541  PMID: 22353253
Cell envelope; Mce operons; Mycobacterium smegmatis; Mycobacterium tuberculosis attenuation
17.  Screening a library of 1,600 adamantyl ureas for anti-Mycobacterium tuberculosis activity in vitro and for better physical chemical properties for bioavailability 
Bioorganic & medicinal chemistry  2012;20(10):3255-3262.
Adamantyl ureas were previously identified as a group of compounds active against Mycobacterium tuberculosis in culture with minimum inhibitor concentrations (MICs) below 0.1 μg/ml. These compounds have been shown to target MmpL3, a protein involved in secretion of trehalose mono-mycolate. They also inhibit both human soluble epoxide hydrolase (hsEH) and M. tuberculosis epoxide hydrolases. However, active compounds to date have high cLogP’s and are poorly soluble, leading to low bioavailability and thus limiting any therapeutic application. In this study, a library of 1,600 ureas (mostly adamantyl ureas), which were synthesized for the purpose of increasing the bioavailability of inhibitors of hsEH, was screened for activity against M. tuberculosis. 1-Adamantyl-3-phenyl ureas with a polar para substituent were found to retain moderate activity against M. tuberculosis and one of these compounds was shown to be present in serum after oral administration to mice. However, neither it, nor a closely related analog, reduced M. tuberculosis infection in mice. No correlation between in vitro potency against M. tuberculosis and the hsEH inhibition were found supporting the concept that activity against hsEH and M. tuberculosis can be separated. Also there was a lack of correlation with cLogP and inhibition of the growth of M. tuberculosis. Finally, members of two classes of adamantyl ureas that contained polar components to increase their bioavailability, but lacked efficacy against growing M. tuberculosis, were found to taken up by the bacterium as effectively as a highly active apolar urea suggesting that these modifications to increase bioavailability affected the interaction of the urea against its target rather than making them unable to enter the bacterium.
doi:10.1016/j.bmc.2012.03.058
PMCID: PMC3345085  PMID: 22522007
adamantyl; urea; tuberculosis; TB drug discovery
18.  Visual Search with Image Modification in Age-Related Macular Degeneration 
Purpose.
AMD results in loss of central vision and a dependence on low-resolution peripheral vision. While many image enhancement techniques have been proposed, there is a lack of quantitative comparison of the effectiveness of enhancement. We developed a natural visual search task that uses patients' eye movements as a quantitative and functional measure of the efficacy of image modification.
Methods.
Eye movements of 17 patients (mean age = 77 years) with AMD were recorded while they searched for target objects in natural images. Eight different image modification methods were implemented and included manipulations of local image or edge contrast, color, and crowding. In a subsequent task, patients ranked their preference of the image modifications.
Results.
Within individual participants, there was no significant difference in search duration or accuracy across eight different image manipulations. When data were collapsed across all image modifications, a multivariate model identified six significant predictors for normalized search duration including scotoma size and acuity, as well as interactions among scotoma size, age, acuity, and contrast (P < 0.05). Additionally, an analysis of image statistics showed no correlation with search performance across all image modifications. Rank ordering of enhancement methods based on participants' preference revealed a trend that participants preferred the least modified images (P < 0.05).
Conclusions.
There was no quantitative effect of image modification on search performance. A better understanding of low- and high-level components of visual search in natural scenes is necessary to improve future attempts at image enhancement for low vision patients. Different search tasks may require alternative image modifications to improve patient functioning and performance.
We examined the effect of image modification on visual search performance in patients with central vision loss from AMD. We found no significant effect on performance measures across eight different image modifications.
doi:10.1167/iovs.12-10012
PMCID: PMC3460389  PMID: 22930725
19.  Safety of and Cellular Response to Segmental Bronchoprovocation in Allergic Asthma 
PLoS ONE  2013;8(1):e51963.
Rationale
Despite its incorporation into research studies, the safety aspects of segmental allergen bronchoprovocation and differences in cellular response among different allergens have received limited consideration.
Methods
We performed 87 segmental challenges in 77 allergic asthma subjects. Allergen dose was based on each subject’s response to whole lung allergen challenge. Bronchoalveolar lavage was performed at 0 and 48 hours. Safety indicators included spirometry, oxygen saturation, heart rate, and symptoms.
Results
Among subjects challenged with ragweed, cat dander, or house dust mite, there were no differences in safety indicators. Subjects demonstrated a modest oxygen desaturation and tachycardia during the procedure that returned to normal prior to discharge. We observed a modest reduction in forced vital capacity and forced expiratory volume in one second following bronchoscopy. The most common symptoms following the procedure were cough, sore throat and fatigue. Total bronchoalveolar lavage fluid cell numbers increased from 13±4 to 106±108×104 per milliliter and eosinophils increased from 1±2 to 44±20 percent, with no significant differences among the three allergens.
Conclusions
In mild allergic asthma, segmental allergen bronchoprovocation, using individualized doses of aeroallergens, was safe and yielded similar cellular responses.
doi:10.1371/journal.pone.0051963
PMCID: PMC3547018  PMID: 23341886
20.  INHIBITION OF MYCOLIC ACID TRANSPORT ACROSS THE MYCOBACTERIUM TUBERCULOSIS PLASMA MEMBRANE 
Nature chemical biology  2012;8(4):334-341.
New chemotherapeutics active against multidrug-resistant Mycobacterium tuberculosis (M. tb) are urgently needed. We report on the identification of an adamantyl urea compound displaying potent bactericidal activity against M. tb and a unique mode of action, namely the abolition of the translocation of mycolic acids from the cytoplasm where they are synthesized to the periplasmic side of the plasma membrane where they are transferred onto cell wall arabinogalactan or used in the formation of virulence-associated outer membrane trehalose-containing glycolipids. Whole genome sequencing of spontaneous resistant mutants of M. tb selected in vitro followed by genetic validation experiments revealed that our prototype inhibitor targets the inner membrane transporter, MmpL3. Conditional gene expression of mmpL3 in mycobacteria and analysis of inhibitor-treated cells validate MmpL3 as essential for mycobacterial growth and support the involvement of this transporter in the translocation of trehalose monomycolate across the plasma membrane.
doi:10.1038/nchembio.794
PMCID: PMC3307863  PMID: 22344175
21.  The Structure Activity Relationship of Urea Derivatives as Anti-Tuberculosis Agents 
Bioorganic & medicinal chemistry  2011;19(18):5585-5595.
The treatment of tuberculosis is becoming more difficult due to the ever increasing prevalence of drug resistance. Thus, it is imperative that novel anti-tuberculosis agents, with unique mechanisms of action, be discovered and developed. The direct anti-tubercular testing of a small compound library led to discovery of adamantyl urea hit compound 1. In this study, the hit was followed up through the synthesis of an optimization library. This library was generated by systematically replacing each section of the molecule with a similar moiety until a clear structure activity relationship was obtained with respect to anti-tubercular activity. The best compounds in this series contained a 1-adamantyl-3-phenyl urea core and had potent activity against Mycobacterium tuberculosis plus an acceptable therapeutic index. It was noted that the compounds identified and the pharmacophore developed is consistent with inhibitors of epoxide hydrolase family of enzymes. Consequently, the compounds were tested for inhibition of representative epoxide hydrolases: M. tuberculosis EphB and EphE; and human soluble epoxide hydrolase. Many of the optimized inhibitors showed both potent EphB and EphE inhibition suggesting the antitubercular activity is through inhibition of multiple epoxide hydrolyase enzymes. The inhibitors also showed potent inhibition of humans soluble expoxide hydrolyase, but limited cytotoxicity suggesting that future studies must be towards increasing the selectivity of epoxide hydrolyase inhibition towards the M. tuberculosis enzymes.
doi:10.1016/j.bmc.2011.07.034
PMCID: PMC3176295  PMID: 21840723
Urea; Tuberculosis; Epoxide Hydrolase
22.  A Phase I/II Study of Erlotinib in Combination with the Anti-Insulin-Like Growth Factor-1 Receptor Monoclonal Antibody IMC-A12 (Cixutumumab) in Patients with Advanced Non-small Cell Lung Cancer 
Journal of Thoracic Oncology  2012;7(2):419-426.
Introduction
This phase I/II study evaluated the safety and anti-tumor effect of the combination of erlotinib with cixutumumab, a recombinant fully humanized anti-insulin-like growth factor-1 receptor IgG1 monoclonal antibody, in advanced non-small cell lung cancer (NSCLC).
Methods
Patients with advanced NSCLC were treated in an initial safety-lead and drop-down cohorts using erlotinib 150 mg/d with cixutumumab 6 or 5 mg/kg on days 1, 8, 15, and 22 in 28-day cycles (cohorts 1 and 2). Emerging pharmacokinetic data led to an additional cohort (3 + 3 design) with cixutumumab at 15 mg/kg on day 1 in 21-day cycles (cohort 3).
Results
Eighteen patients entered the study (6 at 6 mg/kg, 8 at 5 mg/kg, and 4 at 15 mg/kg), with median age of 65 years. Four of six patients at 6 mg/kg experienced dose-limiting toxicities (DLTs), whereas at 5 mg/kg, one of eight patients experienced DLT but three of eight patients still required a dose delay during cycle 1. At 15 mg/kg every 21 days, two of four patients experienced DLTs. In all cohorts, DLTs were either G3 rash or fatigue. Five patients had stable disease as best response and 14 patients had progressive disease. The median progression-free survival was 39 days (range 21–432+ days). Biomarkers analyses showed a trend toward better progression-free survival seen with higher free baseline insulin-like growth factor-1 levels as seen with other insulin-like growth factor-1R inhibitors.
Conclusions
The combinations of cixutumumab at 6 mg/kg every 7 days and 15 mg/kg every 21 days and full-dose erlotinib are not tolerable in unselected patients with NSCLC, as measured by DLT. Cixutumumab at 5 mg/kg every 7 days was tolerable per DLT, but dose delays were common. Efficacy in unselected patients with NSCLC seems to be low.
doi:10.1097/JTO.0b013e31823c5b11
PMCID: PMC3358820  PMID: 22237261
Non-small cell lung cancer; IGF1R monoclonal antibody; EGFR; Metastatic disease
23.  Infliximab treatment of intravenous immunoglobulin-resistant Kawasaki disease 
The Journal of pediatrics  2008;153(6):833-838.
Objective
To test the safety, tolerability, and pharmacokinetics of the anti- TNF-α monoclonal antibody, infliximab, in subjects with intravenous immunoglobulin (IVIG)-resistant Kawasaki disease (KD).
Study design
We conducted a multicenter, randomized, prospective trial of second IVIG infusion (2 g/kg) versus infliximab (5 mg/kg) in 24 children with acute KD and fever following initial treatment with IVIG. Primary outcome measures were infliximab safety, tolerability, and pharmacokinetics. Secondary outcome measures were duration of fever and changes in markers of inflammation.
Results
Study drug infusions were associated with cessation of fever within 24 hours in 11 of 12 subjects treated with infliximab and 8 of 12 subjects retreated with IVIG. No infusion reactions or serious adverse events were attributed to either study drug. No significant differences were observed between treatment groups in the change from baseline for laboratory variables, fever, or echocardiographic assessment of coronary arteries.
Conclusion
Both infliximab and a second IVIG infusion were safe and well-tolerated in subjects with KD who were resistant to standard IVIG treatment. The optimal management of patients resistant to IVIG remains to be determined.
doi:10.1016/j.jpeds.2008.06.011
PMCID: PMC2856847  PMID: 18672254
TNF-α; cytokine; monoclonal antibody; coronary artery aneurysm; infliximab; Kawasaki disease
24.  Role of Porins in the Susceptibility of Mycobacterium smegmatis and Mycobacterium chelonae to Aldehyde-Based Disinfectants and Drugs ▿  
Nosocomial outbreaks attributable to glutaraldehyde-resistant, rapidly growing mycobacteria are increasing. Here, evidence is provided that defects in porin expression dramatically increase the resistance of Mycobacterium smegmatis and Mycobacterium chelonae to glutaraldehyde and another aldehyde disinfectant, ortho-phthalaldehyde. Since defects in porin activity also dramatically increased the resistance of M. chelonae to drugs, there is thus some concern that the widespread use of glutaraldehyde and ortho-phthalaldehyde in clinical settings may select for drug-resistant bacteria.
doi:10.1128/AAC.00590-09
PMCID: PMC2737867  PMID: 19581465
25.  Preliminary crystallographic analysis of GpgS, a key glucosyltransferase involved in methylglucose lipopolysaccharide biosynthesis in Mycobacterium tuberculosis  
Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in Mycobacterium spp. Here, the crystallization and preliminary crystallographic analysis of GpgS from M. tuberculosis and of its complex with UDP are reported.
Glucosyl-3-phosphoglycerate synthase (GpgS) is a key enzyme that catalyses the first glucosylation step in methylglucose lipopolysaccharide biosynthesis in mycobacteria. These important molecules are believed to be involved in the regulation of fatty-acid and mycolic acid synthesis. The enzyme belongs to the recently defined GT81 family of retaining glycosyltransferases (CAZy, Carbohydrate-Active Enzymes Database; see http://www.cazy.org). Here, the purification, crystallization and preliminary crystallographic analysis are reported of GpgS from Mycobacterium tuberculosis and of its complex with UDP. GpgS crystals belonged to space group I4, with unit-cell parameters a = 98.85, b = 98.85, c = 127.64 Å, and diffracted to 2.6 Å resolution. GpgS–UDP complex crystals belonged to space group I4, with unit-cell parameters a = 98.32, b = 98.32, c = 127.96 Å, and diffracted to 3.0 Å resolution.
doi:10.1107/S1744309108032892
PMCID: PMC2593697  PMID: 19052364
glycosyltransferases; methylglucose lipopolysaccharides; Mycobacterium tuberculosis

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